1. Field of the Invention
The present invention relates to the field of computerized image alignment. In particular, the present invention relates to computer systems, methods, and products for aligning grids on scanned images of high-density arrays of biological materials.
2. Related Art
A variety of systems are known for synthesizing or depositing dense arrays of biological materials, sometimes referred to as probes, on a substrate or support. Labeled targets in hybridized probe-target pairs may be detected using various commercial devices, referred to for convenience hereafter as scanners. Scanners image the targets by detecting fluorescent or other emissions from the labels. Data representing the detected emissions are stored in a memory device for processing. The processed images may be presented to a user on a video monitor or other device, and/or operated upon by various data processing products or systems. Some techniques are known for identifying the data representing detected emissions and separating them from background information. For example, U.S. Pat. No. 6,090,555 to Fiekowsky, et al. describes various of these techniques.
The present invention is directed in one embodiment to a method for adjusting the alignment of a grid with an image. Generally speaking, this method seeks an alignment that provides maximum contrast between bright and dim features rather than seeking to identify boundaries, per se, between bright and dim features. The grid in accordance with this method includes grid elements, such as grid squares in an example illustrated below. The image includes image features made up of pixels. For example, the image may include a checkerboard pattern in which the image features are alternating bright and dim squares. Each of the squares is made up of a number of pixels. It will be understood, however, that the pattern is not limited to a checkerboard, and that the image features may be any shape or combination of shapes.
The method includes the step of (1) determining an initial position of the grid. This determination may be made in some implementations such that the grid elements are at least approximately aligned with the plurality of image features. The term approximately is used broadly in this context to mean that, in a typical application, a significant portion of the grid elements are not out of alignment in a particular dimension with respect to image features by more than half the length of the features in that direction. Thus, the grid elements may be significantly out of alignment in one or both dimensions of, for example, a planar grid, but yet be approximately aligned as the term is used herein. It will be understood that although the examples described herein are drawn for convenience to two-dimensional images, the invention is not so limited. Rather, the images and alignment grids may be employed in three or more dimensions.
Additional steps in the method include the following. (2) One or more additional positions of the grid are determined so that each additional position is offset from the initial position and from other additional positions. (3) For each of two or more test grid elements of the two or more grid elements, a set of intensity scores is determined. Each member of this set is based, at least in part, on intensities of pixels of one or more image features within the test grid element in one of the initial or additional positions. (4) For each of the test grid elements, the set of intensity scores is ranked with respect to each other to generate a set of rank scores for the test grid element. (5) For each of the initial and additional grid positions, the members of the sets of rank scores corresponding to the grid position are combined to generate a combined rank score for the grid position. (6) The alignment of the grid is adjusted based, at least in part, on a comparison among the combined rank scores of the initial and additional grid positions.
The first step in this method may be accomplished in accordance with any of variety of known, or yet to be developed, techniques for aligning grids with images. For example, a technique described in U.S. Pat. No. 6,090,555, may be used. The ""555 patent is hereby incorporated by reference herein in its entirety for all purposes. In accordance with one method of the ""555 patent, the image includes a first pattern. Steps in that method include (a) convolving the image with a filter to generate a second image having a second pattern, (b) identifying the second pattern, (c) aligning the grid over the image according to a position of the second pattern, and (d) adjusting the position of the grid to minimize a sum of the intensities of pixels along a direction in the grid. Thus, in this example, steps (a) through (c) implement step (1) of the method described with respect to the present invention.
In some implementations of the method of the present invention, step (2) includes moving the grid from the first grid position in a positive or negative X direction by a value substantially equal to a span of one or more pixels, moving the grid from the first grid position in a positive or negative Y direction by a value substantially equal to a span of one or more pixels, or both. In aspects of these implementations, the test grid elements each have a first length in the X direction and a second length in the Y direction. Step (2) in these aspects further includes moving the grid from the first grid position in the positive or negative X direction by a value no greater than approximately half the first length, moving the grid from the first grid position in the positive or negative Y direction by a value no greater than approximately half the second length, or both.
Step (3) may include determining, for at least one member of the set of intensity scores, a mean of intensity values of pixels within the test grid element. Also, step (3) may include the following two steps: (a) based on the initial grid position, identifying each of the plurality of grid elements as either a dim grid element or, in the alternative, a bright grid element; and (b) selecting the test grid elements based, at least in part, on including dim grid elements when they are bordered on all sides by a bright grid element. In other implementations, the dim grid elements may be including among the test grid elements if they are bordered on fewer than all sides, or on no side, by a bright grid element. Some bright grid elements may also not be included among the test grid elements in some implementations.
In some aspects of the preceding method, step (3)(a) may include the further steps of (i) based on a determined pattern (such as, for example, a checkerboard), associating each of the plurality of grid elements with one of either a first element group or a second element group; (ii) determining a first intensity of one or more of the grid elements in the first element group and a second intensity of one or more of the grid elements in the second element group; (iii) comparing the first and second intensities; (iv) designating the grid elements in the first element group as dim grid elements when the first intensity is less than the second intensity and as bright grid elements when the first intensity is greater than the second intensity; and (v) designating the grid elements in the second element group as dim grid elements when the second intensity is less than the first intensity and as bright grid elements when the second intensity is greater than the first intensity. Step (3) (a)(ii) may include determining a median of intensities of three or more pixels, or every pixel, within each of the one or more grid elements in the first and second element groups. Similarly, in other aspects, step (3)(a)(ii) may include determining a median of intensities of three or more pixels, or every pixel, within each grid element in the first and second element groups. The first intensity may include a first overall median of the medians of intensities of pixels within each grid element in the first element group. The second intensity may include a second overall median of the medians of intensities of pixels within each grid element in the second element group.
In some implementations, step (4) of the method of the present invention may further include the step of ranking the sets of intensity scores of test grid elements identified as dim grid elements in an opposite sense to the ranking of sets of intensity scores of test grid elements identified as bright grid elements. For example, grid elements determined to be bright elements may be ranked by sorting their intensities in order of decreasing intensity with the element having the brightest intensity score ranked lowest (e.g., 1). Grid elements determined to be dim elements may be ranked by sorting their intensities in order of increasing intensity with the element having the dimmest intensity score ranked lowest (e.g.,
Another embodiment of the present invention is directed to an image alignment tool for adjusting the alignment of a grid with an image. The grid includes a plurality of grid elements and the image includes a plurality of image features comprised of pixels. The image alignment tool includes an initial grid aligner that determines an initial position of the grid. Also included is a grid offsetter that determines one or more additional positions of the grid that each are offset from the initial position and from each other. An intensity scorer also is included that, for each of a plurality of test grid elements of the plurality of grid elements, determines a set of intensity scores. Each member of the set is based, at least in part, on intensities of pixels of one or more image features within the test grid element in one of the initial or additional positions. Another element of the tool is an intensity-score ranker that, for each of the test grid elements, ranks the set of intensity scores with respect to each other to generate a set of rank scores for the test grid element. Yet another element is a combined-rank-score generator that, for each of the initial and additional grid positions, combines the members of the sets of rank scores corresponding to the grid position to generate a combined rank score for the grid position. The image alignment tool of this embodiment also includes a grid alignment adjuster that adjusts the alignment of the grid based, at least in part, on a comparison among the combined rank scores of the initial and additional grid positions.
The invention is directed in yet further embodiments to a computer program product for adjusting the alignment of a grid with an image. The computer program product typically is embodied in a computer-readable medium, such as a CD-ROM, a disk, or in internal RAM or other memory. Moreover, the product may be implemented in other forms, such as firmware or a combination of firmware and software. The grid includes grid elements, and the image includes image features made up of pixels. The method includes the following steps. (1) An initial position of the grid is determined such that the grid elements are at least approximately aligned with the plurality of image features. (2) One or more additional positions of the grid are determined so that each additional position is offset from the initial position and from other additional positions. (3) For each of two or more test grid elements of the two or more grid elements, a set of intensity scores is determined. Each member of this set is based, at least in part, on intensities of pixels of one or more image features within the test grid element in one of the initial or additional positions. (4) For each of the test grid elements, the set of intensity scores is ranked with respect to each other to generate a set of rank scores for the test grid element. (5) For each of the initial and additional grid positions, the members of the sets of rank scores corresponding to the grid position are combined to generate a combined rank score for the grid position. (6) The alignment of the grid is adjusted based, at least in part, on a comparison among the combined rank scores of the initial and additional grid positions.
In another embodiment, the invention is directed to a computer system for processing images. The computer system includes a data storage device that stores data representing an image that has image features comprised of pixels. The computer system also has an image alignment tool, in accordance with the present invention, for adjusting the alignment of a grid with the image. In a yet further embodiment, the invention is directed to a scanning system that includes an excitation source that produces emissions from labeled targets hybridized to probe features, and a detector that detects the emissions and generates data representing one or more characteristics of the emissions. The scanning system also includes a data storage device that stores an image having image features comprised of pixels based on data generated by the detector. Also included in the scanning system is an image alignment tool, in accordance with the present invention, for adjusting the alignment of a grid with the image.
A method is also described for analyzing an array of probes including biological polymers. The method includes: (a) contacting the array with targets including biological polymers under conditions effective for binding between the targets and the probes; (b) detecting an association between the targets and probes using an imaging system to produce data that represents the association, the data being capable of forming an image; (c) associating the data with a first set of locations on the array; (d) analyzing the data to determine a measure of contrast; and (e) re-associating the data with a second set of locations based, at least in part, on the measure of contrast. The above embodiments are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, aspect of the invention. The description of one embodiment is not intended to be limiting with respect to other embodiments. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative embodiments, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiments are illustrative rather than limiting.